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High-tamp. resistant threonine synthetase gene, polypeptide coded therewith and preparing method thereof

A technology of threonine and high temperature resistance, which is applied in the field of mutation or genetic engineering, and can solve the problem that the function of threonine synthase has not been reported.

Inactive Publication Date: 2003-05-28
HUADA GENE RES & DEV CENT HANGZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There is no report about the function of threonine synthase other than synthesis

Method used

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  • High-tamp. resistant threonine synthetase gene, polypeptide coded therewith and preparing method thereof
  • High-tamp. resistant threonine synthetase gene, polypeptide coded therewith and preparing method thereof
  • High-tamp. resistant threonine synthetase gene, polypeptide coded therewith and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Construction of a sequencing library

[0035] The sequencing library was constructed using the genome-wide shotgun method. Firstly, Tengchong thermophilic anaerobic bacteria were cultured according to (Yanfen Xue, 2000) modified MB medium (Balch et al., 1979), bacteria were collected according to Marmur (1961) method, and total DNA was extracted. In order to ensure the randomness of sequencing library construction and avoid the problem of hot spots of breakage to the greatest extent, a variety of methods and principles of library construction under different conditions were adopted. First, physical shearing methods (including ultrasonic method and HydroshearMachine shearing) were used, and then AluI was selected for random partial enzyme digestion according to the genome characteristics of the bacteria. Different intensities were used to process samples during physical shearing, and samples were processed by setting enzyme gradients during enzyme digestion. ...

Embodiment 2

[0036] Example 2: Genome Sequencing

[0037] When sequencing the genome of thermophilic anaerobic bacteria in Tengchong, two automatic sequencers were mainly used: ABI377 and MegaBACE 1000. Both sequencers use the principle of electrophoresis for sequencing (see figure 2 ), 96 samples can be completed each time. ABI377 is a product of PE Company, which is a kind of ABI series. It belongs to the slab gel electrophoresis sequencer. MegaBACE 1000 is a product of Pharmacia, which belongs to capillary gel electrophoresis sequencer.

Embodiment 3

[0038] Example 3: Basecalling and sequencing quality monitoring

[0039] The so-called basecalling refers to the process of obtaining the correct base sequence from the raw data file obtained on the sequencer. Since the sequencer obtains the intensity change traces (traces) of light of different wavelengths corresponding to the four bases A, T, G, and C, it is necessary to use a computer to adopt a certain algorithm to correctly identify the bases corresponding to the different traces. . We used Phred software (Ewing B, Hillier L, 1998) because its results are more reliable and its output is more convenient for further analysis by other programs in the same software package.

[0040] The algorithm principle of Phred's basecalling is to judge the base type based on the shape, distance, and signal-to-noise ratio of each peak in the trajectory, and at the same time give the credibility information for this base, that is, the sequencing quality of the base. In large-scale sequen...

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Abstract

A process for coding the separated DNA with activity or its function equivalent variant and preparing the polypeptide with high-temp resistant threonine synthetase activity or its function equivalentvariant from said DNA by recombination is disclosed. The high-temp resistant threonine synthetase gene is cloned and separated, which can be used to prepare the transgenic microbe, animal or plant and recover the enzyme coded by said gene. The amino acid sequence of said polypeptide and the processes for preparing, separating and purifying the polypeptide are also disclosed.

Description

technical field [0001] The invention relates to mutation or genetic engineering, in particular to a gene sequence of a high-temperature-resistant threonine synthetase, a coded polypeptide and a preparation method. Background technique [0002] Threonine synthase (Threonine synthase, EC 4.2.99.2) catalyzes the reaction: [0003] L-homoserine phosphate + water = threonine + inorganic phosphate In microorganisms, the synthesis of threonine is an initial part of the multi-branched biosynthetic pathway, starting to synthesize aspartic acid, lysine, and methylsulfide amino acid, isoleucine. Threonine synthase is a PLP (pyridoxal-5-phosphate)-dependent enzyme that catalyzes the last step in threonine biosynthesis, catalyzing L-homoserine phosphate (HSerP) to threonine and inorganic phosphate Salt. In this PLP-dependent β,γ-displacement reaction, the enzyme-acted substrate forms Schiff and the enzyme-binding factor PLP, followed by...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N9/88C12N15/52C12N15/60C12N15/63C12N15/64
Inventor 包其郁杨焕明卢小羽胡松年刘斯奇
Owner HUADA GENE RES & DEV CENT HANGZHOU